The present invention relates to a chemical compound that has applications as a luminescent indicator dye, and to an optical sensor, typically employed for determination of near-neutral pH values of aqueous samples. The optical sensor has particular application in the pH determination of body liquids such as, for example, blood, plasma and serum.
Measuring the pH is an essential task in many fields of science and technology, for instance in chemistry, process engineering, manufacturing and environmental analysis. A number of optical sensors for determination of pH have been proposed. Surveys with emphasis on determination and monitoring of blood pH by means of optical sensors have been given by Leiner and Wolfbeis, Fiber Optic pH Sensors, in CRC BOOK ON FIBER OPTIC CHEMICAL SENSORS AND BIOSENSORS (O. S. Wolfbeis ed., CRC Press Inc., Boca Raton, Fla. (1991)) and Leiner and Hartmann, Theory and practice in optical pH sensing, SENSORS AND ACTUATORS, B 11, 281-289 (1993).
For blood gas analysis it is essential that pH is determined very accurately. See Leiner, Optical sensors for in vitro blood gas analysis, SENSORS AND ACTUATORS, B 29, 169-173 (1995).
Recently, new optical sensors suitable for measurement of sodium and potassium in serum, plasma and whole blood samples have been described. The optical sensors are based on PET dyes immobilized in a hydrophilic polymer layer. See He et al., A fluorescent chemosensor for sodium based on photoinduced electron transfer, ANAL. CHEM. 75, 549-555 (2003); He et al., A fluorescent sensor with high selectivity and sensitivity for potassium in water, J. AM. CHEM. SOC. 125, 1468-1469 (2003). The “PET effect” (PET=photoinduced electron transfer) denotes the photone induced transfer of electrons from a donor to luminophoric moiety.
PET dyes sensitive to pH are known, which dyes were initially used to study luminescent PET systems (Bissel et al., Luminescence and Charge Transfer. Part 2. Aminomethyl Anthracene Derivatives as Fluorescent PET (Photoinduced Electron Transfer) Sensors for Protons, J. CHEM. SOC. PERKN TRANS 2, 1559-1564 (1992)) in solvents. In early studies, aliphatic and aromatic amines were suggested as the pH sensitive part (donor part) for the PET dye. The latter were attached to luminescent polycyclic aromatic compounds (acceptor part) to yield a pH sensitive PET dye. PET dyes containing amino-groups show a strong difference in luminescence intensity of protonated and deprotonated species. The donor part bound via a spacer group to the acceptor part acts as a luminescence quencher. In the protonated state no quenching of the luminescence of the electronically excited acceptor part occurs. In the deprotonated state, the PET donor group quenches the luminescence of the electronically excited acceptor part. The quenching efficiency depends on the ability of the quencher part to transfer an electron to the electronically excited acceptor part and on the ability of the electronically excited acceptor part to accept the electron.
Since the ratio of protonated and de-protonated dye species depends on both the pH (pH=−log(concentration or activity of protons)) in the vicinity of the PET donor group and the pK of the pH-sensitive chemical group of the PET donor part, luminescence intensity of the PET dye depends on pH. The pK is defined as pH at which the ratio of protonated and de-protonated dye species equals 1.
In general, the useful pH-range, i.e., the pH range, where significant changes of luminescence intensity occur is about pK+/−1.5 units.
For determination of near neutral pHs of watery samples it is therefore required that the pK of the PET donor group is near neutral (close to 7) in an aqueous environment. For determination of blood pH at 37° C. by means of an optical sensor the 37° C. pK measured by exposing the sensor to calibration solutions of different pHs, is most preferably close to 7.4+/−0.3 pH units.
Preferred amines for PET quenching are unsubstituted aliphatic amines (i.e., —CH2—NH2; NOT —CH2—NRH or —CH2—NR2) and unsubstituted aromatic amines (i.e., phenyl-NH2). Typical pKs of unsubstitutes aliphatic amines are near 9. Typical pKs of unsubstituted aromatic amines are near 3-4, the exact pK depending on the specific chemical environment and the temperature.
It is further required that the dye part possesses favorable absorbance (preferably higher then 450 nm) and emission wavelengths (preferably higher then 500 nm).
It is further required that the dye part is insensitive to notoric quenchers like oxygen. The latter is in particular not the case for oxygen-sensitive dyes (i.e., transition metal complexes).
As water-soluble dyes present in a hydrophilic matrix are generally easily washed out by aqueous samples, it is generally required to attach the dye to the matrix, most preferably by covalent linking. Dyes containing chemical groups for covalent attachment, i.e., via chemical reactions under mild ambient conditions are preferred.
Moreover, it is most advantageous that—within the pH range of interest—the dye part of a PET indicator dye is essentially insensitive to pH. Thus, for example, the fluorescein dye has a protonable group with a pK within the near neutral pH range.
Werner et al. (Novel optical pH-sensor based on a borodiaza-indacene derivative, FRESENIUS J. ANAL. CHEM. 359, 150-154 (1997)) describe a pH sensor based on a PET dye (1,3,5,7-tertramethyl-8-(4-dimethylamino)-4-difluorobora-3a,4a-diaza(s)-indacene) immobilized in a hydrogel matrix. The quencher group is dimethyl amino phenyl. The aromatic nitrogen reversibly reacts with protons. Due to its low pK (pK=3.3; see FIG. 3 in Werner et al., supra), the luminescence intensity of this indicator dye changes as a function of pH within the pH-range (˜1.5-4.5). Accordingly, the dye is not useful for determination of physiol. pHs (i.e., blood).
Gareis et al. (Phenol/phenolate-dependent on/off switching of the luminescence of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes, CHEM. COMMUN. 1717-1718 (1997)) reported that a 4-difluorobora-3a,4a-diaza(s)-indacene with a phenolic quencher group (PET donor group) shows a strong PET effect.
Wolfbeis et al., describe a number of PET dyes with —NR2 and —OH functional groups for determination of pH (see U.S. Pat. No. 6,001,999, col. 4, FIG. 2. and claim 8).
A titration of the dye (Gareis et al., supra) dissolved in CHCl3 showed a strong decrease of the 520 nm emission band upon successive addition of pyridine. Gareis et al. embedded the dye in a hydrogel matrix of an optical sensor. In the matrix, the base form of the dye (phenolate species) showed low luminescence intensity, whereas the acid form of the indicator dye (phenol species) showed high luminescence intensity. From pH titration curves of the two phenols investigated, the pKs were determined to be 10.4 and 10.8, respectively.